NK cells provide a first line of defense against a variety of microbial antigens and are potent producers of cytokines that modulate the response of many other cell types. In addition, experimental and clinical studies highlight the potential for NK cells to enhance antitumor responses. However, relatively little is known about the positive regulatory signaling events generated by the novel activating receptors on NK cells. In fact, recent published work and preliminary data presented in this application suggest that the current paradigm for signaling in T cells and B cells is insufficient to explain signal transduction in NK cells. As an alternative model, we hypothesize that: (a) NKG2D/DAP10- and 2B4-initiated killing are regulated in a Src-family tyrosine kinase-dependent, but Syk-family independent manner; (b) the tyrosine in the DAP10 YINM consensus motif regulates not only its association with PI3-kinase, but also additional PTK-dependent signals regulating NK cell activation; (c) the 2B4 complex on activated NK cells is associated with one (or more) Src-family tyrosine kinases; (d) although stimulation of DAP10 or 2B4 alone on NK cells is sufficient to drive a cellular killing response, DAP10 and 2B4 can functionally amplify a simultaneous ITAM-initiated signal; (e) Rac3 is a strong positive regulator of NK cell-mediated cytotoxicity; (f) Rac-stimulated PAK1 and Rho-stimulated ROCK together synergistically upregulate LIM-kinase activity and the subsequent actin cytoskeleton changes that immediately precede granule exocytosis and killing; and (g) dynamin 2 regulates the development of NK cell-mediated cytotoxicity through complementary GTPase-dependent and GTPase independent mechanisms. In order to test these hypotheses we will (1) Evaluate the differential signaling pathways utilized by the novel NK cell activating receptors NKG2D/DAP10 and 2B4; (2) Identify and characterize specific regulatory roles for the Rac/Rho family GTPases and their effectors in NK cell activation; and (3) Determine the mechanisms by which the GTPase dynamin 2 controls the generation of NK cell-mediated cytotoxicity. Together these studies will provide an experimental basis for understanding the molecular events that are involved in the regulation of NK cell effector functions, and will, in a broader context, advance our understanding of fundamental processes involved in transmembrane signaling and lymphocyte activation.